1. Field of the Invention
The present invention relates to an induced pluripotent stem cell (iPS) model for cardiofaciocutaneous (CFC) syndrome, a method for producing the model, and uses of the iPS model in the analysis of neural development in CFC syndrome.
2. Description of the Related Art
Cardiofaciocutaneous syndrome is a genetic disease accompanied by heart deformity, unique facial shape, abnormal skin condition, and retarded development. The heart deformity is exemplified by valvar pulmonary stenosis, ventricular or atrial septal defect, and hypertrophic cardiomyopathy. The unique facial shape is characterized by comparatively big head, frontal bossing, thin eyebrows, and exophthalmos. The abnormal skin is exemplified by xerosis, hyperkeratosis, ichthyosis, eczema, and loose scalp (Roberts A et al. J Med Genet (2006) 43:833-42; Armour C M and Allanson J E. J Med Genet (2008) 45:249-54). The clinical symptoms of CFC syndrome are similar to those of Noonan syndrome and thus CFC syndrome is classified into the Noonan-associated disease together with Costello syndrome and LEOPARD syndrome.
It was recently reported that RAS-MAPK (mitogen-activated protein kinase) signaling pathway associated genes including PTPN11, HRAS, KRAS, BRAF, MEK1, and MEK2 are the major cause of Noonan syndrome and Noonan-associated di seases. The mutation of those genes above causes cell growth and differentiation associated symptoms mediated by the stimulation of a growth factor. Studies on the detection and diagnosis of Noonan syndrome and Noonan-associated diseases have been focused on the screening of the gene mutation above (Jorge A A et al. Horm Res (2009) 71:185-93). However, it has not been confirmed that CFC syndrome is directly associated with the mutation of the said genes and accordingly a cause or a treatment method has not been explained, yet.
To understand Noonan syndrome and Noonan-associated syndrome, various approaches have been made. As a result, it was reported that p-ERK generated by the phosphorylation of ERK protein in the RAS-MAPK signaling pathway was up-regulated in Noonan syndrome, NF1 syndrome, and LEOPARD syndrome (T Nakamura et al. PNAS (2009), 106:36, 15436-15441; Yuan Wang et al. cell (2012) 150:4 816-830; X Carvajal-Vergara et al. Nature (2010) 465, 808-812). However, when a drug directly targeting p-ERK was used as a treating agent for Noonan syndrome and Noonan-associated syndrome, the signaling pathway was directly blocked and most likely various side effects were expected in relation to cell proliferation or cell differentiation. Therefore, it is requested to develop or to screen a drug that can indirectly inhibit p-ERK.
Stem cell is the cell in the pre-differentiation phase toward the differentiation into each cell forming different tissues, which can be obtained from each tissue of an embryo, a fetus, and an adult and characteristically exhibits self-proliferating activity enabling unlimited proliferation in undifferentiated state and pluripotency that is the potential for the differentiation into various tissues once a specific differentiation stimulus is given. Stem cell is differentiated into a specific cell by a differentiation stimulus (environment). Unlike the differentiated cell whose cell division is arrested, stem cell is self-renewal and has the ability of proliferation (expansion) and plasticity, meaning it can produce the cell that is the same as itself and at the same time it can be differentiated into different cells when the environment is changed or a different stimulus is given.
Human pluripotent stem cells including induced pluripotent stem cells (hPSCs) including induced pluripotent stem cells (iPS) have the capability of being differentiated into specific but various cell kinds. So, when induced pluripotent stem cells (iPS) are used in in vitro differentiation system, not only therapeutic potential including lowering the risk of immunorejection is increased but also the mechanism of complex disease in the early stage of organogenesis can be easily understand, making them as an efficient evaluator (Muotri, A. R. (2009) Epilepsy Behav 14 Suppl 1: 81-85; Marchetto, M. C., B. Winner, et al. (2010) Hum Mol Genet 19(R1): R71-76).
Therefore, the present inventors tried to establish a stem cell model for the research of CFC syndrome. The inventors induced the development and differentiation of CFC syndrome-derived induced pluripotent stem cells (iPS) into embryonic body from the fibroblasts of a CFC syndrome patient and then confirmed that the CFC syndrome-derived iPS and embryonic body exhibited broken embryonic body shapes and no differentiation into neurons. The inventors also induced CFC syndrome-derived embryonic body by treating with p-ERK and p-SMAD1 inhibitors. As a result, the embryonic body displayed a normal embryonic body shape and was effectively differentiated into neurons. Therefore, the method for cell modeling of CFC syndrome using the iPS of the invention can be efficiently used for the screening of CFC syndrome treating agents, leading to the completion of the invention.